Methods and compositions for oxygen transport comprising modified hemoglobin in plasma

ABSTRACT

The present invention relates to blood products, and more particularly to compositions comprising a modified hemoglobin in plasma.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a 371 of PCT/US03/00695 filed Jan. 10, 2003, whichclaims the priority of the provisional application No. 60/347,739 filedon Jan. 11, 2002.

TECHNICAL FIELD

The present invention relates to blood substitutes, and moreparticularly to compositions comprising a modified hemoglobin in plasma.

BACKGROUND OF THE INVENTION

The Circulatory System and the Nature of Hemoglobin

The blood is the means for delivering nutrients to the tissues andremoving waste products from the tissues for excretion. The blood iscomposed of plasma in which red blood cells (RBCs or erythrocytes),white blood cells (WBCs), and platelets are suspended. Red blood cellscomprise approximately 99% of the cells in blood, and their principalfunction is the transport of oxygen to the tissues and the removal ofcarbon dioxide therefrom.

The left ventricle of the heart pumps the blood through the arteries andthe smaller arterioles of the circulatory system. The blood then entersthe capillaries, where the majority of the exchange of nutrients andcellular waste products occurs. (See, e.g., A. C. Guyton, HumanPhysiology And Mechanisms Of Disease (3rd. ed.; W. B. Saunders Co.,Philadelphia, Pa.), pp. 228-229 (1982)). Thereafter, the blood travelsthrough the venules and veins in its return to the right atrium of theheart. Though the blood that returns to the heart is oxygen-poorcompared to that which is pumped from the heart, when at rest, thereturning blood still contains about 75% of the original oxygen content.

The reversible oxygenation function (i.e., the delivery of oxygen) ofRBCs is carried out by the protein hemoglobin. In mammals, hemoglobinhas a molecular weight of approximately 64,000 daltons and is composedof about 6% heme and 94% globin. In its native form, it contains twopairs of subunits (i.e., it is a tetramer), each containing a heme groupand a globin polypeptide chain. In aqueous solution, hemoglobin ispresent in equilibrium between the tetrameric (MW 64,000) and dimericforms (MW 32,000); outside of the RBC, the dimers are prematurelyexcreted by the kidney (plasma half-life of approximately 2-4 hours).Along with hemoglobin, RBCs contain stroma (the RBC membrane), whichcomprises proteins, cholesterol, and phospholipids.

Exogenous Blood Products

Due to the demand for blood products in hospitals and other settings,extensive research has been directed at the development of bloodsubstitutes and plasma expanders. A blood substitute is a blood productthat is capable of carrying and supplying oxygen to the tissues. Bloodsubstitutes have a number of uses, including replacing blood lost duringsurgical procedures and following acute hemorrhage, and forresuscitation procedures following traumatic injury. Plasma expandersare blood substitutes that are administered into the vascular system butare typically not capable of carrying oxygen. Plasma expanders can beused, for example, for replacing plasma lost from burns, to treat volumedeficiency shock, and to effect hemodilution (e.g., for the maintenanceof normovolemia and to lower blood viscosity). Essentially, bloodsubstitutes can be used for these purposes or any purpose in whichbanked blood is currently administered to patients. (See, e.g., U.S.Pat. No. 4,001,401 to Bonson et al., and U.S. Pat. No. 4,061,736 toMorris et al.)

The current human blood supply is associated with several limitationsthat can be alleviated through the use of an exogenous blood substitute.To illustrate, the widespread availability of safe and effective bloodsubstitutes would reduce the need for banked (allogeneic) blood.Moreover, such blood substitutes would allow the immediate infusion of aresuscitation solution following traumatic injury without regard tocross-matching (as is required for blood), thereby saving valuable timein resupplying oxygen to ischemic tissue. Likewise, blood substitutescan be administered to patients prior to surgery, allowing removal ofautologous blood from the patients which could be returned later in theprocedure, if needed, or after surgery. Thus, the use of exogenous bloodproducts not only protects patients from exposure to non-autologous(allogeneic) blood, it conserves either autologous or allogeneic(banked, crossmatched) blood for its optimal use.

Limitations of Current Blood Substitutes

Attempts to produce blood substitutes (sometimes referred to as“oxygen-carrying plasma expanders”) have thus far produced products withmarginal efficacy or whose manufacture is tedious and expensive, orboth. Frequently, the cost of manufacturing such products is so highthat it effectively precludes the widespread use of the products,particularly in those markets where the greatest need exists (e.g.,emerging third-world economies).

Blood substitutes can be grouped into the following three categories: i)perfluorocarbon-based emulsions, ii) liposome-encapsulated hemoglobin,and iii) modified cell-free hemoglobin. As discussed below, none hasbeen entirely successful, though products comprising modified cell-freehemoglobin are thought to be the most promising. Perfluorochemical-basedcompositions dissolve oxygen as opposed to binding it as a chelate. Inorder to be used in biological systems, the perfluorochemical must beemulsified with a lipid, typically egg-yolk phospholipid. Though theperfluorocarbon emulsions are inexpensive to manufacture, they do notcarry sufficient oxygen at clinically tolerated doses to be effective.Conversely, while liposome-encapsulated hemoglobin has been shown to beeffective, it is far too costly for widespread use. (See generally,Winslow, Robert M., “Hemoglobin-based Red Cell Substitutes”, JohnsHopkins University Press, Baltimore, 1992).

Most of the blood substitute products in clinical trials today are basedon modified hemoglobin. These products, frequently referred to ashemoglobin-based oxygen carriers (HBOCs), generally comprise ahomogeneous aqueous solution of a chemically-modified hemoglobin,essentially free from other red cell residue (stroma). Althoughstroma-free hemoglobin (SFH) from humans is the most common raw materialfor preparing a HBOC, other sources of hemoglobin have also been used.For example, hemoglobin can be obtained or derived from animal blood(e.g., bovine or porcine hemoglobin) or from bacteria or yeast ortransgenic animals molecularly altered to produce a desired hemoglobinproduct.

The chemical modification is generally one of intramolecularcross-linking, oligomerization and/or polymer conjugation to modify thehemoglobin such that its persistence in the circulation is prolongedrelative to that of unmodified hemoglobin, and its oxygen bindingproperties are similar to those of blood. Intramolecular cross-linkingchemically binds together subunits of the tetrameric hemoglobin unit toprevent the formation of dimers which, as previously indicated, areprematurely excreted. (See, e.g., U.S. Pat. No. 5,296,465 to Rausch etal.)

The high costs of manufacturing HBOC products have greatly limited theircommercial viability. In addition, the present inventors have found thatknown HBOCs have a tendency to release excessive amounts of oxygen tothe tissues at the arteriole walls rather than the capillaries. This canresult in insufficient oxygen available for delivery by the HBOC to thetissues surrounding the capillaries. This is despite the fact that theinitial loading of the HBOC with oxygen may be relatively high, evenhigher than that normally achieved with natural red blood cells.

In addition, most blood substitutes under development are limited toHBOCs in colloid solutions and solutions having relatively lowosmolarity. (See, e.g., U.S. Pat. Nos. 5,814,601 and 5,661,124). Whilesuch mixtures are sufficient for some blood replacement uses, anenhancement of the therapeutic effects of the blood substitute isdesired for other uses. Such enhancement can be provided by supplyingthe HBOC in plasma. This provides the dual benefit of enhancing thedelivery of oxygen to the tissues and providing the benefit of plasmareplacement. Accordingly, the present invention relates to a bloodsubstitute that comprises HBOC in plasma.

SUMMARY OF THE INVENTION

The present invention relates to the use of surface-modified hemoglobinsin plasma as blood substitutes. The plasma may be from a natural source,and is preferably from the same animal species as the hemoglobin. Morepreferably, the plasma is autologous plasma, i.e., it comes from therecipient themselves. Other aspects of the present invention aredescribed throughout the specification.

DESCRIPTION OF THE INVENTION

The present invention is directed to blood substitutes comprising HBOCsand plasma. For certain applications, there is a synergistic effect ofenhancing oxygen delivery using HBOCs and administering plasma.

Definitions

To facilitate understanding of the invention set forth in the disclosurethat follows, a number of terms are defined below.

The term “hemoglobin” refers generally to the protein contained withinred blood cells that transports oxygen. Each molecule of hemoglobin has4 subunits, 2 α chains and 2 β chains, which are arranged in atetrameric structure. Each subunit also contains one heme group, whichis the iron-containing center that binds oxygen. Thus, each hemoglobinmolecule can bind 4 oxygen molecules.

The term “modified hemoglobin” includes, but is not limited to,hemoglobin altered by a chemical reaction such as intra- andinter-molecular cross-linking, genetic manipulation, polymerization,and/or conjugation to other chemical groups (e.g., polyalkylene oxides,for example polyethylene glycol, or other adducts such as proteins,peptides, carbohydrates, synthetic polymers and the like). In essence,hemoglobin is “modified” if any of its structural or functionalproperties have been altered from its native state. As used herein, theterm “hemoglobin” by itself refers both to native, unmodified,hemoglobins as well as modified hemoglobin.

The term “surface-modified hemoglobin” is used to refer to hemoglobindescribed above to which chemical groups such as dextran or polyalkyleneoxide have been attached, most usually covalently.

The term “stroma-free hemoglobin” refers to hemoglobin from which allred blood cell membranes have been removed.

The term “perfluorocarbons” refers to synthetic, inert, molecules thatcontain fluorine atoms, and that consist entirely of halogen (Br, F, Cl)and carbon atoms. In the form of emulsions, they are under developmentas blood substances, because they have the ability to dissolve manytimes more oxygen than equivalent amounts of plasma or water.

The term “plasma expander” refers to any solution that may be given to asubject to treat blood loss.

The term “oxygen carrying capacity”, or simply “oxygen capacity” refersto the capacity of a blood substitute to carry oxygen, but does notnecessarily correlate with the efficiency in which it delivers oxygen.Oxygen carrying capacity is generally calculated from hemoglobinconcentration, since it is known that each gram of hemoglobin binds 1.34ml of oxygen. Thus, the hemoglobin concentration in g/dl multiplied bythe factor 1.34 yields the oxygen capacity in ml/dl. Hemoglobinconcentration can be measured by any known method, such as by using theB-Hemoglobin Photometer (HemoCue, Inc., Angelholm, Sweden). Similarly,oxygen capacity can be measured by the amount of oxygen released from asample of hemoglobin or blood by using, for example, a fuel cellinstrument (e.g., Lex-O₂-Con; Lexington Instruments).

The term “oxygen affinity” refers to the avidity with which an oxygencarrier such as hemoglobin binds molecular oxygen. This characteristicis defined by the oxygen equilibrium curve which relates the degree ofsaturation of hemoglobin molecules with oxygen (Y axis) with the partialpressure of oxygen (X axis). The position of this curve is denoted bythe value, P50, the partial pressure of oxygen at which the oxygencarrier is half-saturated with oxygen, and is inversely related tooxygen affinity. Hence the lower the P50, the higher the oxygenaffinity. The oxygen affinity of whole blood (and components of wholeblood such as red blood cells and hemoglobin) can be measured by avariety of methods known in the art. (See, e.g., Winslow et al., J.Biol. Chem. 252(7):2331-37 (1977)). Oxygen affinity may also bedetermined using a commercially available HEMOX™ TM Analyzer (TCSScientific Corporation, New Hope, Pa.). (See, e.g., Vandegriff andShrager in Methods in Enzymology (Everse et al., eds.) 232:460 (1994)).

The terms “hypertonic” and “hyperosmolar” means an osmolarity greaterthan 800 mOsm/l, which is the average osmolarity of whole blood. Thephrase “highly hypertonic” refers to solutions with an osmolaritygreater than 2000 mOsm/l. Osmolarity may be measured by any suitabletechnique, such as in a Wescor instrument (Ontario, Canada).

The term “oxygen-carrying component” refers broadly to a substancecapable of carrying oxygen in the body's circulatory system anddelivering at least a portion of that oxygen to the tissues. Inpreferred embodiments, the oxygen-carrying component is native ormodified hemoglobin, and is also referred to herein as a “hemoglobinbased oxygen carrier”, or “HBOC”.

The term “hemodynamic parameters” refers broadly to measurementsindicative of blood pressure, flow and volume status, includingmeasurements such as blood pressure, cardiac output, right atrialpressure, and left ventricular end diastolic pressure.

The term “crystalloid” refers to small molecules (usually less than 10Å) such as salts, sugars, and buffers. Unlike colloids, crystalloids donot contain any oncotically active components and therefore leave thecirculation very quickly.

The term “colloid”, in contrast to “crystalloid” refers to largermolecules (usually greater than 10 Å) that do not freely pass throughbiological membranes and includes proteins such as albumin and gelatin,as well as starches such as pentastarch and hetastarch.

The term “colloid oncotic pressure” or “colloid osmotic pressure” refersto the propensity of colloids to remain in the interavascular space forprolonged periods of time drawing water from the interstitial andintracellular spaces into the intravascular space.

Finally, the term “mixture” refers to a mingling together of two or moresubstances without the occurrence of a reaction by which they would losetheir individual properties; the term “solution” refers to a liquidmixture; the term “aqueous solution” refers to a solution that containssome water and may also contain one or more other liquid substances withwater to form a multi-component solution; the term “approximately”refers to the actual value being within a range, e.g. 10%, of theindicated value. The meaning of other terminology used herein should beeasily understood by someone of reasonable skill in the art.

The Nature of Oxygen Delivery and Consumption

Although the successful use of the compositions and methods of thepresent invention do not require comprehension of the underlyingmechanisms of oxygen delivery and consumption, basic knowledge regardingsome of these putative mechanisms may assist in understanding thediscussion that follows. It has generally been assumed that thecapillaries are the primary conveyors of oxygen to the tissue. However,regarding tissue at rest, current findings indicate that there isapproximately an equipartition between arteriolar and capillary oxygenrelease. That is, hemoglobin in the arterial system is believed todeliver approximately one third of its oxygen content in the arteriolarnetwork and one-third in the capillaries, while the remainder exits themicrocirculation via the venous system.

The arteries themselves are sites of oxygen utilization. For example,the artery wall requires energy to effect regulation of blood flowthrough contraction against vascular resistance. Thus, the arterial wallis normally a significant site for the diffusion of oxygen out of theblood. However, current oxygen-delivering compositions (e.g., HBOCs) mayrelease too much of their oxygen content in the arterial system, andthereby induce an autoregulatory reduction in capillary perfusion.Accordingly, the efficiency of oxygen delivery of a blood substitute mayactually be hampered by having too much oxygen or too low an oxygenaffinity.

The rate of oxygen consumption by the vascular wall, i.e., thecombination of oxygen required for mechanical work and oxygen requiredfor biochemical synthesis, can be determined by measuring the gradientat the vessel wall. See, e.g., Winslow, et al., in “Advances in BloodSubstitutes” (1997), Birkhauser, ed., Boston, Mass., pages 167-188.Present technology allows accurate oxygen partial pressure measurementsin a variety of vessels. The measured gradient is directly proportionalto the rate of oxygen utilization by the tissue in the region of themeasurement. Such measurements show that the vessel wall has a baselineoxygen utilization which increases with increases in inflammation andconstriction, and is lowered by relaxation.

The vessel wall gradient is inversely proportional to tissueoxygenation. Vasoconstriction increases the oxygen gradient (tissuemetabolism), while vasodilation lowers the gradient. Higher gradientsare indicative of the fact that more oxygen is used by the vessel wall,while less oxygen is available for the tissue. The same phenomenon isbelieved to be present throughout the microcirculation.

Oxygen Carrying Component

In preferred embodiments, the oxygen carrier (i.e., the oxygen-carryingcomponent) is a hemoglobin-based oxygen carrier, or HBOC. The hemoglobinmay be either native (unmodified); subsequently modified by a chemicalreaction such as intra- or inter-molecular cross-linking,polymerization, or the addition of chemical groups (e.g., polyalkyleneoxides, or other adducts); or it may be recombinantly engineered. Humanalpha- and beta-globin genes have both been cloned and sequenced.Liebhaber, et al., P.N.A.S. 77: 7054-7058 (1980); Marotta, et al., J.Biol. Chem. 353: 5040-5053 (1977) (beta-globin cDNA). In addition, manyrecombinantly produced modified hemoglobins have now been produced usingsite-directed mutagenesis, although these “mutant” hemoglobin varietieswere reported to have undesirably high oxygen affinities. See, e.g.,Nagai, et al., P.N.A.S. 82: 7252-7255 (1985).

The present invention is not limited by the source of the hemoglobin.For example, the hemoglobin may be derived from animals and humans.Preferred sources of hemoglobin are humans, cows and pigs. In addition,hemoglobin may be produced by other methods, including chemicalsynthesis and recombinant techniques. The hemoglobin can be added to theblood product composition in free form, or it may be encapsulated in avesicle, such as a synthetic particle, microballoon or liposome. Thepresent invention also contemplates the use of other means for oxygendelivery that do not entail hemoglobin or modified hemoglobin, such asthe fluorocarbon emulsions.

The preferred oxygen-carrying components of the present invention shouldbe stroma free and endotoxin free. Representative examples ofoxygen-carrying components are disclosed in a number of issued UnitedStates Patents, including U.S. Pat. No. 4,857,636 to Hsia; U.S. Pat. No.4,600,531 to Walder, U.S. Pat. No. 4,061,736 to Morris et al.; U.S. Pat.No. 3,925,344 to Mazur; U.S. Pat. No. 4,529,719 to Tye; U.S. Pat. No.4,473,496 to Scannon; U.S. Pat. No. 4,584,130 to Bocci et al; U.S. Pat.No. 5,250,665 to Kluger et al.; U.S. Pat. No. 5,028,588 to Hoffman etal.; and U.S. Pat. No. 4,826,811 and U.S. Pat. No. 5,194,590 to Sehgalet al.

However, as discussed above, the present inventors theorize that bloodsubstitutes with lower oxygen affinities may trigger autoregulatoryevents that prevent oxygen delivery to the tissues via microcapillarycirculation. Accordingly, using the experimental models described inWinslow, supra, it has been determined that, for some applications anHBOC with an oxygen affinity less than that of SFH is desired. Thisfinding is contrary to conventional teachings in the field.

There are many different scientific approaches to manufacturing HBOCswith high oxygen affinity (i.e. those with P50s less than SFH). Forexample, studies have identified the amino acid residues that play arole in oxygen affinity, and thus site-directed mutagenesis can now beeasily carried out to manipulate oxygen affinity to a desired level.See, e.g., U.S. Pat. No. 5,661,124. Many other approaches are discussedin U.S. Pat. No. 6,054,427.

Modifications of the Oxygen-Carrying Component

In a preferred embodiment, the oxygen-carrying component is modifiedhemoglobin. A preferred modification to hemoglobin is“surface-modification”, i.e. covalent attachment of chemical groups tothe exposed amino acid side chains on the hemoglobin molecule. Mostcommonly, the chemical group attached to the hemoglobin is polyethyleneglycol (PEG), because of its pharmaceutical acceptability and commercialavailability. PEGs are polymers of the general chemical formulaH(OCH₂CH₂)_(n)OH, where n is greater than or equal to 4. PEGformulations are usually followed by a number that corresponds to theiraverage molecular weight. For example, PEG-200 has an average molecularweight of 200 and may have a molecular weight range of 190-210. PEGs arecommercially available in a number of different forms, and in manyinstances come preactivated and ready to conjugate to proteins.

The number of PEGs to be added to the hemoglobin molecule may vary,depending on the size of the PEG. However, the molecular size of theresultant modified hemoglobin should be sufficiently large to avoidbeing cleared by the kidneys to achieve the desired half-life.Blumenstein, et al., determined that this size is achieved above 84,000molecular weight. (Blumenstein, et al., in “Blood Substitutes and PlasmaExpanders”, Alan R. Liss, editors, New York, N.Y., pages 205-212(1978).) Therein, the authors conjugated hemoglobin to dextran ofvarying molecular weight. They reported that a conjugate of hemoglobin(with a molecular weight of 64,000) and dextran (having a molecularweight of 20,000) “was cleared slowly from the circulation andnegligibly through the kidneys”, but increasing the molecular weightabove 84,000 did not alter the clearance curves. Accordingly, asdetermined by Blumenstein, et al., it is preferable that the HBOC have amolecular weight of at least 84,000.

Plasma Component

The blood substitutes of the present invention further comprise plasma,preferably animal plasma, and more preferably human plasma. Although notwishing to be bound by any particular scientific theory, it is believedthat the administration of blood substitutes may dilute theconcentration of coagulation factors to an undesirable level.Accordingly, using plasma as the diluent for the oxygen carryingcomponent avoids this problem. Plasma can be collected by any meansknown in the art, provided that red cells, white cells and platelets areessentially removed. Preferably, it is obtained using an automatedplasmaphoresis apparatus. Plasmaphoresis apparatuses are commerciallyavailable and include, for example, apparatuses that separate plasmafrom the blood by ultrafiltration or by centrifugation. Anultrafiltration-based plasmaphoresis apparatus such as manufactured byAuto C, A200 (Baxter International, Largo, Fla.) is suitable because iteffectively removes red cells, white cells and platelets whilepreserving coagulation factors.

Plasma may be collected with an anticoagulant, many of which are wellknown in the art. Preferred anti-coagulants are those that chelatecalcium such as citrate. Sodium Citrate at 0.38% (final concentration inthe plasma) is the preferred anticoagulant for collecting plasma. Theplasma may be fresh, frozen, pooled and/or sterilized.

In addition to plasma from natural sources, it is contemplated thatsynthetic plasma is also included within the present invention, andincludes any aqueous solution that is at least isotonic and that furthercomprises at least one plasma protein.

While plasma from exogenous sources may be preferred, it is also withinthe present invention to use autologous plasma that is collected fromthe subject prior to formulation and administration of the bloodsubstitute.

Crystalloid Component

In one embodiment of the present invention, the blood substitute mayalso comprise a crystalloid. The crystalloid component can be anycrystalloid which, in the form of the blood substitute composition, ispreferably capable of achieving an osmolarity greater than 800 mOsm/l,i.e. it makes the blood substitute “hypertonic”. Examples of suitablecrystalloids and their concentrations in the blood substitute include,e.g., 3% NaCl, 7% NaCl, 7.5% NaCl, and 7.5% NaCl in 6% dextran. Morepreferably, the blood substitute has an osmolarity of between 800 and2400 mOsm/l. The use of recombinantly produced hemoglobins in solutionswith an osmolality between 300-800 mOsm/l that further comprise acolloid (i.e. a molecule less diffusible than dextrose) have beenpreviously reported. See, e.g., U.S. Pat. No. 5,661,124. However, thispatent teaches away from producing blood substitutes with osmolalitiesabove 800, and suggests that the hemoglobin concentration should bebetween 6-12 g/dl. In contrast, the oxygen carrying efficiency ofcompositions of the present invention permit lower concentrations ofhemoglobin to be used, such as less than 6 g/dl or even less than 4g/dl.

When the blood substitute further comprises a crystalloid and ishypertonic, the compositions of present invention may provide improvedfunctionality for rapid recovery of hemodynamic parameters over otherblood substitute compositions, which include a colloid component. Smallvolume highly hypertonic crystalloid infusion (e.g., 1-10 ml/kg)provides significant benefits in the rapid and sustained recovery ofacceptable hemodynamic parameters in controlled hemorrhage. (See, e.g.,Przybelski, R. J., E. K. Daily, and M. L. Birnbaum, “The pressor effectof hemoglobin—good or bad?” In Winslow, R. M., K. D. Vandegriff, and M.Intaglietta, eds. Advances in Blood Substitutes. IndustrialOpportunities and Medical Challenges. Boston, Birkhauser (1997), 71-85).Hypertonic crystalloid solutions alone, however, do not adequatelyrestore cerebral oxygen transport. See D. Prough, et al., Effects ofhypertonic saline versus Ringer's solution on cerebral oxygen transportduring resuscitation from hemorrhagic shock J. Neurosurg. 64:627-32(1986).

Formulation

The blood substitutes of the present invention are formulated by mixingthe oxygen carrier and other optional excipients with a suitable diluentthat is at least 40% plasma. Although the concentration of the oxygencarrier in the diluent may vary according to the application, and inparticular based on the expected post-administration dilution, inpreferred embodiments, because of the other features of the compositionsof the present invention that provide for enhanced oxygen delivery andtherapeutic effects, it is usually unnecessary for the concentration tobe above 6 g/dl, and is more preferably between 0.1 to 4 g/dl.

Clinical Applications

The methods and compositions of the present invention are useful in avariety of different applications, such as hemodilution, trauma, septicshock, ischemia, cancer, anemia, cardioplegia, hypoxia and organperfusion. These and other applications are discussed extensively inU.S. Pat. No. 6,054,427.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inhematology, surgical science, transfusion medicine, transplantation, orany related fields are intended to be within the scope of the followingclaims.

1. A blood substitute composition comprising polyethylene glycolsurface-modified hemoglobin having an oxygen affinity less than that ofstroma-free hemoglobin, and an aqueous diluent, wherein the aqueousdiluent is at least 40% plasma.
 2. The blood substitute composition ofclaim 1, wherein the plasma is derived from a natural source.
 3. Theblood substitute composition of claim 1, wherein the plasma is derivedfrom the same animal species as the hemoglobin.
 4. The blood substitutecomposition of claim 1, wherein the plasma is autologous plasma.
 5. Theblood substitute composition of claim 1, wherein the hemoglobinconcentration is less than 4g/dL.